1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58and black tea Part 1: Content of total polyphenols in tea Colorimetric method using Folin-Ciocalteu
2、 reagentICS 67.140.10 Determination of substances characteristic of green BRITISH STANDARDBS ISO 14502-1:2005Incorporating Corrigendum No. 1 BS ISO 14502-1:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 12 April 2005 BSI 2006ISBN 0 580 4
3、5778 8 The British Standards which implement international publications referred to enquiries on the interpretation, or proposals for change, and keep in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search
4、” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard does not of itself confer immunity from legal obliga
5、tions.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 10, an inside back cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publica
6、tionAmd. No. Date Comments16367Corrigendum No. 131 July 2006 Correction to Figure A.1A list of organizations represented on this committee can be obtained on request to its secretary.Cross-referencesUK interests informed; monitor related international and European developments and promulgate them in
7、 the UK.National forewordThis British Standard reproduces verbatim ISO 14502-1:2005, including Technical Corrigendum April 2006 and implements it as the UK national standard.The UK participation in its preparation was entrusted to Technical Committee AW/8, Tea, which has the responsibility to: aid e
8、nquirers to understand the text; present to the responsible international/European committee any Reference numberISO 14502-1:2005(E)INTERNATIONAL STANDARD ISO14502-1First edition2005-03-01Determination of substances characteristic of green and black tea Part 1: Content of total polyphenols in tea Co
9、lorimetric method using Folin-Ciocalteu reagent Dtermination des substances caractristiques du th vert et du th noir Partie 1: Dosage des polyphnols totaux dans le th Mthode colorimtrique utilisant le ractif de Folin-Ciocalteu BS ISO 145021:2005ii BS ISO 145021:2005iiiContents Page Foreword iv 1 Sco
10、pe 1 2 Normative references . 1 3 Principle . 1 4 Reagents 1 5 Apparatus. 3 6 Sampling 3 7 Preparation of test samples. 3 8 Procedure. 4 8.1 General. 4 8.2 Determination of dry matter content. 4 8.3 Test portion . 4 8.4 Extraction of polyphenols 4 8.5 Dilution. 5 8.6 Determination 5 9 Calculation. 5
11、 10 Precision 6 10.1 Interlaboratory test . 6 10.2 Repeatability 6 10.3 Reproducibility 6 11 Test report 7 Annex A (informative) Gallic acid calibration graph . 8 Annex B (informative) Results of interlaboratory test 9 Bibliography . 10 BS ISO 145021:2005iv Foreword ISO (the International Organizati
12、on for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established ha
13、s the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Inte
14、rnational Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publica
15、tion as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rig
16、hts. ISO 14502-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 8, Tea. ISO 14502 consists of the following parts, under the general title Determination of substances characteristic of green and black tea: Part 1: Content of total polyphenols in tea Colorimetric method
17、 using Folin-Ciocalteu reagent Part 2: Content of catechins in green tea Method using high-performance liquid chromatography BS ISO 145021:20051Determination of substances characteristic of green and black tea Part 1: Content of total polyphenols in tea Colorimetric method using Folin-Ciocalteu reag
18、ent 1 Scope This part of ISO 14502 specifies a method for the determination of the total polyphenol content of leaf tea and instant tea by a colorimetric assay using Folin-Ciocalteu phenol reagent 4. It is applicable to both green and black tea products. 2 Normative references The following referenc
19、ed documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 1572, Tea Preparation of ground sample of known dry matter content
20、ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 7513, Instant tea in solid form Determination of moisture content (loss in mass at 103 C) 3 Principle Polyphenols are extracted with 70 % methanol from a test portion of finely ground leaf tea at 70 C. Instant teas
21、 are dissolved in hot water with 10 % acetonitrile (volume fraction) added to stabilize the extract. The polyphenols in the extract are determined colorimetrically using Folin-Ciocalteu phenol reagent. The reagent contains phospho-tungstic acids as oxidants, which on reduction by readily oxidized ph
22、enolic hydroxy groups yield a blue colour with a broad maximum absorption at 765 nm. This is due to the formation of so-called tungsten and molybdenum blues. The Folin-Ciocalteu phenol reagent reacts with a wide range of polyphenol compounds and, although the response can vary with the individual co
23、mponents, selection of gallic acid as a calibration standard enables useful total polyphenol data to be obtained. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 4.1 Water, conforming to grade 1 of ISO 3696:1987. 4.2 Acetonitrile. 4.3 Methanol. BS ISO 145021:
24、20052 4.4 Methanol/water extraction mixture, 70 % methanol (volume fraction). Add 700 ml of the methanol (4.3) to a 1 litre one-mark volumetric flask. Dilute to the mark with water (4.1) and mix. 4.5 Folin-Ciocalteu phenol reagent, commercially available ready prepared. It is advisable to check the
25、calibration linearity with respect to gallic acid in order to ascertain the suitability of the supplied reagent. 4.6 Dilute Folin-Ciocalteu phenol reagent, 10 % (volume fraction). Using a pipette, transfer 20 ml of Folin-Ciocalteu phenol reagent (4.5) to a 200 ml one-mark volumetric flask. Dilute to
26、 the mark with water and mix. Prepare fresh reagent solution daily. To avoid contamination of the concentrated Folin-Ciocalteu reagent, discard any unused dispensed reagent. 4.7 Sodium carbonate solution, 7,5 % (mass concentration). Weigh (37,50 0,01) g of anhydrous sodium carbonate (Na2CO3) into a
27、500 ml one-mark volumetric flask. Add sufficient warm water to half-fill the flask. Swirl to dissolve the sodium carbonate, cool to room temperature, dilute to the mark with water and mix. NOTE This solution will remain stable at room temperature for up to 1 month. 4.8 Gallic acid stock standard sol
28、ution, corresponding to approximately 1 000 g/ml of anhydrous gallic acid. Weigh (0,110 0,001) g of gallic acid monohydrate (M = 188,14) into a 100 ml one-mark volumetric flask. Dissolve in water, dilute to the mark and mix. Prepare a fresh standard solution daily. NOTE Gallic acid monohydrate is pr
29、eferred over anhydrous, due to its greater solubility, reduced hygroscopic properties and availability of certified reagent grades, e.g. A.C.S., which is used to denote chemicals that meet specifications outlined by the American Chemical Society. If not known, the moisture content (loss in mass at 1
30、03 C) on a portion of the standard material should be determined. The concentration of the stock standard solution on a gallic acid anhydrous basis can then be calculated. 4.9 Gallic acid standard solutions A to E Using pipettes, transfer the volumes of gallic acid stock standard solution (4.8) give
31、n in Table 1 to 100-ml one-mark volumetric flasks. Dilute to the mark with water and mix. These dilute standard solutions should be prepared on the day of use. Table 1 Gallic acid dilute standard solutions Gallic acid standard solution Volume of gallic acid stock solution Nominal concentration of di
32、lute standard ml g/ml A 1,0 10 B 2,0 20 C 3,0 30 D 4,0 40 E 5,0 50 BS ISO 145021:200535 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Analytical balance, capable of weighing to an accuracy of 0,001 g. 5.2 Water bath, capable of being maintained at 70 C 1 C. 5.3 Dispense
33、r, for methanol/water extraction mixture (4.4), and set at 5,0 ml. 5.4 Centrifuge, capable of 2 000 Relative Centrifugal Force (R.C.F.) (typically 3 500 r/min). 5.5 Spectrophotometer, set at 765 nm and able to accommodate 10 mm path length cells, preferably in an automatic flow cell assembly. 5.6 Pi
34、pettes, glass or automatic, to cover the volume range for standard and sample extract dilutions. 5.7 One-mark volumetric flasks, of capacities 100 ml, 200 ml, 500 ml, and 1 litre. 5.8 Vortex mixer, for efficient mixing during extraction. 5.9 Extraction tubes, glass, of 10 ml capacity, stoppered and
35、able to withstand being centrifuged. 5.10 Graduated tubes, glass, of 10 ml capacity with 0,1 ml graduations. As the assay is sensitive to traces of organic impurities, extraction tubes (5.9) and graduated tubes (5.10) should all be taken through an additional cleaning procedure of washing in approx.
36、 15 % (volume fraction) nitric acid, followed by rinsing thoroughly in water and drying in an air oven at 100 C. The use of disposable plastic tubes as an alternative to the graduated tubes in the final colorimetic assay is recommended, as additional cleaning has not been found necessary. 6 Sampling
37、 A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this part of ISO 14502. A recommended sampling method is given in ISO 1839 for leaf tea, and ISO 7516 for instant
38、tea. 7 Preparation of test samples To ensure homogeneity, grind the sample of leaf tea in accordance with ISO 1572 and store samples in well-sealed containers protected from light. Grinding of instant tea is only required on samples of a coarse granular structure. BS ISO 145021:20054 8 Procedure 8.1
39、 General If it is required to check whether the repeatability limit (10.2) is met, carry out two single determinations in accordance with 8.2 to 8.6 under repeatability conditions. 8.2 Determination of dry matter content Calculate the dry matter content from the moisture content (loss in mass at 103
40、 C) determined on a portion of the test sample (Clause 7) in accordance with ISO 1572 for leaf tea, or ISO 7513 for instant tea. 8.3 Test portion 8.3.1 Instant tea Weigh (0,500 0,001) g of the test sample (Clause 7) into a 50 ml one-mark volumetric flask. 8.3.2 Leaf tea Weigh (0,200 0,001) g of the
41、test sample (Clause 7) into an extraction tube (5.9). 8.4 Extraction of polyphenols 8.4.1 Instant tea 8.4.1.1 Add to the instant tea in the flask (8.3.1) approximately 25 ml of hot water (maximum temperature 60 C). Mix to dissolve the sample and allow to cool to room temperature. 8.4.1.2 Add 5,0 ml
42、of acetonitrile (4.2). Dilute to the mark with water and mix. 8.4.2 Leaf tea 8.4.2.1 Place the methanol/water extraction mixture (4.4) contained in the dispenser (5.3) in the water bath (5.2) set at 70 C, and allow at least 30 min for the extraction mixture to equilibrate. 8.4.2.2 Place the extracti
43、on tube containing the sample (8.3.2) in the water bath set at 70 C. Dispense 5,0 ml of hot methanol/water extraction mixture from 8.4.2.1 into the extraction tube, stopper and mix on the vortex mixer (5.8). 8.4.2.3 Continue heating the extraction tube in the water bath for 10 min, mixing on the vor
44、tex mixer after 5 min and 10 min. It is important to mix the samples thoroughly to ensure complete extraction. 8.4.2.4 Remove the extraction tube from the water bath and allow it to cool to room temperature. Remove the stopper and place the tube in the centrifuge (5.4) at 3 500 r/min for 10 min. 8.4
45、.2.5 Carefully decant the supernatant into a graduated tube (5.10). 8.4.2.6 Repeat extraction steps 8.4.2.2 to 8.4.2.5. Combine the extracts, make up to 10 ml with cold methanol/ water extraction mixture (4.4) and mix the contents. BS ISO 145021:200558.4.2.7 The extract from 8.4.2.6 is stable for at
46、 least 24 h if stored at 4 C. Allow the extract to attain room temperature before proceeding with the assay. Resuspension of the small amount of particulate material that may settle during storage is not necessary. 8.5 Dilution Using a pipette, transfer 1,0 ml of the sample extract (instant tea extr
47、act from 8.4.1.2 or leaf tea extract from 8.4.2.6) into a one-mark 100 ml volumetric flask. Dilute to the mark with water and mix. 8.6 Determination 8.6.1 Using a pipette, transfer 1,0 ml of the gallic acid standard solutions A, B, C, D and E (4.9), in duplicate, into separate plastic or graduated t
48、ubes (5.10). NOTE These correspond to approximately 10 g, 20 g, 30 g, 40 g and 50 g of anhydrous gallic acid. 8.6.2 Using a pipette, transfer 1,0 ml of water, in duplicate, into separate tubes (5.10). These are reagent blanks. 8.6.3 Using a pipette, transfer 1,0 ml of diluted sample extract (8.5), i
49、n duplicate, into separate tubes. 8.6.4 Using a pipette, add 5,0 ml of dilute Folin-Ciocalteu phenol reagent (4.6) into each tube and mix. 8.6.5 Within 3 min to 8 min after the addition of the dilute Folin-Ciocalteu phenol reagent, pipette 4,0 ml of sodium carbonate solution (4.7) into each tube. Stopper and mix. 8.6.6 Allow to stand at room temperature for 60 min, and then measure the optical densities in 10-mm path length cells against water on the spectr
